1. Field of the Invention
The present invention relates to a method of cross-linking peptides through a novel amino acid sequence Q-X-K-(S/T) (SEQ ID NO: 15), which was initially identified from mouse seminal vesicle secretion (SVS) III protein. The peptides containing Q-X-K-(S/T) (SEQ ID NO: 15) sequence may be cross-linked by any transgluatminase (TGase).
2. Description of the Related Art
Cross-linking of biomolecules has been noted for decades throughout the world of biotechnology. Cross-linking of proteins with different functions can produce a new molecule with multi functions. Enzymes cross-link to a solid phase makes its activity retaining that can reduce manufactory costs. Biomolecules can be cross-linked by chemical reactions while these reactions are not very specific and probably reduced the activity of enzymes. An alternative way to cross-link biomolecules is enzyme-catalyzed reaction.
Transgluatminase (TGase) is a kind of enzyme with the ability to catalyze protein-protein cross-linking reaction. Transglutaminase (TGase) was first reported by Heinrich Waelsch in 1959. He isolated the enzyme from the liver of guinea pig. In the presence of calcium ion, this enzyme shows trans-amidation activity which binding glutamine in proteins to primary amines covalently. Therefore this enzyme was designed as transglutaminase (EC.2.3.2.13). In recent years, more transglutaminases have been purified from different spices and different tissues. Until now, TGases can be divided into five types: (1) tissue type TGase (TG2), which is commonly expressed in all kinds of tissues, might involve cell-programmed death; (2) epidermal TGase (TG1), found in the wounded epidermal tissues, enable epidermal proteins to cross-link into keratin; (3) hair-follicle TGase, found in hair-follicle cell, can cross-link hair protein; (4) plasma factor XIII, when catalyzed by thrombin, it can cross-link fibrin to stabilize the structure of thrombus; (5) prostate TGase (TG4), found in the coagulating gland of rodent, can catalyze seminal vesicle secreted proteins to copulatory plug. Although these different kinds of TGases have great difference in size and sequences, their catalytic mechanisms are similar. Each of them has a cystine residue in their active site and the enzyme activity is calcium dependent. TGase has great usages in industry. For examples: adding TGase to meat will increase its tenacity and savor in food processing; Enzymes can be fixed by cross-linking of TGase in enzyme engineering; TGase has been used to construct tissues' frame in tissue engineering et cetera.
However, there are limits in industrial applications for TGase. First, most TGase was isolated from animal source and they are also difficult to prepare by recombinant techniques. Second, most TGases have specificities to their substrates that limits the application of TGase. J. E. Folk and his group made a series of studies on tissue type TGase of guinea pig's liver and human's plasma factor XIII (Gorman, J. J. and Folk, J. E. 1980. J. Biol. Chem. 2255, 4419-427; Schhrode, J. and Folk, J. E. 1979. J. Biol. Chem. 254, 653-661). Their works may help to understand the substrate specificity of TGase. TGase has two substrates: one is usually defined as glutamine in a peptides chain and serves as an acyl donor; the other should be a primary amine, which serves as an acyl acceptor. TGases demand more specificity of acyl donor than of acyl acceptor in usual. Therefore, lots of polyamines (spermine and histamine for example) also can be acyl acceptors and covalently bind with proteins as a kind of post-translational modification.
Some works have been done to define the effective sites from substrates of TGases to produce cross-linking peptides fragment. For example, in U.S. Pat. No. 5,428,014 and U.S. Pat. No. 5,939,385, peptide sequences from human plasma fibrinogen have been studied. These peptide fragments are proved to be cross-linkable by human plasma factor XIII and this characteristic has been applied in tissue engineering. This invention generalized an S1-Y-S2 formula from plasma fibrinogen. In this case, S1 represents T-I-G-E-G-Q (SEQ ID NO: 10), Y is 0˜7 interval amino acids, and S2 is X-K-X-A-G-D-V (SEQ ID NO: 11) (U.S. Pat. No. 5,428,014, claim 1). Yet, this invention didn't define characteristics and effects of the amino acids in Y position. Moreover, the peptide fragments in this invention were only effective under the action of human plasma factor XIII that limits the usage of other sources of TGases and also restricts the utilities of these peptide fragments. Besides, the length of the defined fragment was too long and the reaction efficiency was low.
In the present invention, we have found a better substrate of TGase from other sources. Seminal vesicles secretions of rodent have been reported as good substrate of TGase (Notides, A. C. and Williams-Ashman, H. G. 1967. Proc. Natl. Acad. Sci. U.S.A. 58, 1991-1995). Notides and Williams-Ashman found a protein (18 kDa) secreted from guinea pig's seminal vesicle. This protein can readily be polymerized by a TGase secreted from coagulating gland. Following study also proved that SVS II protein from mouse and rat seminal vesicle secretions are substrates of TGase (Harris, S. E. et. al. 1990. J. Biol. Chem. 265, 9896-9903; Lundwall, A. et al. 1997. Eur. J. Biochem. 249, 39-44). Though human seminal secretions will not solidify to become copulatory plugs, it has been proved that SgI and SgII proteins from human seminal vesicle are also substrates of TGase (Peter, A. et. Al. 1998. Eur. J. Biochem. 252, 216-221). However, the molecular mechanism of these proteins have never been studied. In this invention, we isolated a new protein, SVS III, from mouse seminal vesicle and proved it a good substrate of transglutaminase. The present invention also provides an effective sequence from SVS III and related applications.